The heavy metal cadmium is a widespread environmental contaminant that accumulates in the body and poses a threat to human health. Bone is a critical target site for cadmium. Human exposure to cadmium is linked to bone diseases, including osteoporosis and osteopenia. In 2005, a CDC report listed cadmium as a toxin that merits monitoring. This report, combined with the escalating cost of healthcare for osteoporosis, emphasizes the importance of promoting research to decipher the underlying mechanisms of cadmium-induced osteotoxicity. Despite its recognized importance as an environmental toxin, little is known about how cadmium directly impacts bone cells, in particular the bone-forming osteoblasts. We previously reported that cadmium induces apoptosis, programmed cell death, in human osteoblast-like cells (Saos-2). The goal of this research is to determine the intracellular mechanisms involved in cadmium-induced apoptosis using an in vitro osteoblast cell model. Evidence indicates that oxidative stress, enhanced generation of reactive oxygen species (ROS), plays a role in cadmium toxicity and the pathogenesis of osteoporosis. Specific aim 1) is to investigate the role of oxidative stress (e.g., ROS formation, glutathione depletion, and lipid peroxidation) in cadmium-induced osteoblast apoptosis. The transcriptional factor RUNX2 is a known critical mediator of the osteoblast phenotype and research indicates RUNX2 plays a protective role against osteoporosis in postmenopausal women. Therefore, specific aim 2) is to determine if cadmium-induced oxidative stress leads to a decrease in RUNX2 expression. The methods employed include RT-PCR and Western blot analysis to detect changes in RUNX2 mRNA and protein expression, respectively. Cells will be treated with antioxidants to prevent cadmium-induced apoptosis and decrease in RUNX2 expression. Lastly, key to understanding the mechanisms involved in cadmium-induced apoptosis is deciphering the signaling pathways involved. Specific aim 3) is to explore the role of the protein kinase C (PKC) pathway in cadmium-induced osteoblast apoptosis. The methods used include using known PKC inhibitors to block cadmium-induced decrease in RUNX2 expression and apoptosis. Confirmation of PKC involvement will be done by assessing active phosphorylated forms of PKC by Western blot analysis. This research will provide insight into mechanism underlying cadmium-induced disruption of apoptotic signaling in bone. In turn, this cadmium osteotoxicity model may help identify targets for treatment and prevention of osteoporosis. The long term goal is to develop an experimental animal model using adult zebrafish to validate these in vitro studies and link cadmium-induced osteoblast apoptosis to net bone loss. PUBLIC HEALTH RELEVANCE: The heavy metal cadmium, a widespread environmental contaminant and component in cigarettes, accumulates in the body and poses a threat to human health. Bone is a critical target site for cadmium and human exposure to cadmium is linked to bone disease, including osteoporosis. This research is relevant because it provides insight into how cadmium directly impacts bone, and may help identify targets for treatment and prevention of osteoporosis. [unreadable] [unreadable] [unreadable]